SUMMARY Over 3 billion people worldwide are affected by herpes simplex virus type 1 (HSV-1), a highly successful neurotropic virus that resides in the nervous system. Although most infected immunocompetent individuals do not develop any herpetic disease, a small number of mostly immunosuppressed individuals are at risk of developing neuro-pathogenesis and life-threatening Herpes Simplex Encephalitis (HSE). Mortality from HSE appears to be caused by aberrant immune responses in the central nervous system (CNS), rather than direct virus cytopathology. The efficacy of antiviral drugs to treat human encephalitis remains unproven. Our long- term goal is to develop an alternative immunotherapeutic strategy. While antiviral CD8+ T cells appear to protect from herpes infection and disease, major gaps in our understanding still remain, including: (1) the relative contributions of various CD8+ T cell subpopulations (i.e., CD8+ effector memory (TEM), tissue-resident memory (TRM) cells, and central memory (TCM) cells) in protecting against HSE; and (2) how virulent HSV-1 strains interfere with the antiviral function of trigeminal ganglia- (TG-) and CNS-resident HSV-specific CD8+ T cells. Our preliminary results using a well-established ?humanized? HLA transgenic mouse model of HSE indicate that infection with the most neurovirulent HSV-1 strains (e.g., McKrae and KOS 79) induced high levels of CD8+ T cell exhaustion (but not CD4+ T cell exhaustion) that expressed high levels of PD-1, TIM-3, and LAG-3 exhaustion markers, and that this was associated with CNS inflammation and HSE symptoms that led to death in ~30% of animals. In contrast, in less virulent HSV-1 strains (e.g., KOS), the presence of a high number of functional anti-viral CD8+ T cells in the CNS and sensory ganglion was associated with reduced CNS inflammation and death. Building on these preliminary results, we hypothesize that HSE results from an increase in T cell exhaustion and dysregulated CNS- and TG-resident T cell function induced by neurovirulent HSV-1 strains. We will test this hypothesis through two Specific Aims: Aim 1: Confirm the hypothesis that following infection with a virulent HSV-1 laboratory strain (i.e., McKrae) or with a virulent HSV- 1 clinical isolate (i.e., KOS 79), a high frequency of dysfunctional (exhausted) HSV-specific CNS- and TG- resident CD8+ T cells, expressing high levels of PD-1, TIM-3, and LAG-3 exhaustion markers, is associated with HSE. Aim 2: Test the hypothesis that single or combined blockade of PD-1, TIM-3, and LAG-3 immune checkpoints will induce more functional CNS- and TG-resident anti-viral CD8+ T cells and result in more robust and sustained protection against HSE following infection of HLA Tg mice with a virulent HSV-1 strain. Successful completion of this project, which covers both mechanistic (Aim 1) and translational research (Aim 2), should guide the development of novel blockade of T cell immune checkpoints to restore HSV-specific T cells? effector functions against neuro-pathogenesis and life-threatening Herpes Simplex Encephalitis.